The Multifaceted Profile of Thyroid Disease in the Background of DICER1 Germline and Somatic Mutations: Then, Now and Future Perspectives

DICER1 protein is a member of the ribonuclease (RNAse) III family with a key role in the biogenesis of microRNAs (miRNA) and in microRNA processing, potentially affecting gene regulation at the post-transcriptional level. The role of DICER1 and its relevance to thyroid cellular processes and tumorigenesis have only recently been explored, following the acknowledgement that DICER1 germline and somatic changes can contribute not only to non-toxic multinodule goiter (MNG) lesions detected in individuals of affected families but also to a series of childhood tumours, including thyroid neoplasms, which can be identified from early infancy up until the decade of 40s. In a context of DICER1 germline gene mutation, thyroid lesions have recently been given importance, and they may represent either an index event within a syndromic context or the isolated event that may trigger a deeper and broader genomic analysis screening of individuals and their relatives, thereby preventing the consequences of a late diagnosis of malignancy. Within the syndromic context MNG is typically the most observed lesion. On the other hand, in a DICER1 somatic mutation context, malignant tumours are more common. In this review we describe the role of DICER protein, the genomic events that affect the DICER1 gene and their link to tumorigenesis as well as the frequency and pattern of benign and malignant thyroid lesions and the regulation of DICER1 within the thyroidal environment.

Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer

In complex biochemical systems, an enzyme, protein, or RNA, symbolized as E, has hundreds or thousands of substrates or interacting partners. The relative specificity hypothesis proposes that such an E would differentially interact with and influence its many distinct, downstream substrates, thereby regulating the underlying biological process (es). The importance of relative specificity has been underappreciated, and evidence of its physiological consequences particularly lacking. Previously we showed that human Drosha and Dicer ribonucleases (RNases) both discriminate their respective microRNA (miRNA) substrates, and that differential cleavage by Drosha contributes to global differential miRNA expression. If relative specificity is an important biological mechanism, it should be evolutionarily conserved. To test this hypothesis, we hereby examined the cleavage of hundreds of zebrafish and fruitfly miRNA intermediates by Drosha and Dicer and the impact on miRNA biogenesis in these organisms. We showed that Drosha action regulates differential miRNA expression in zebrafish and fruitflies and identified the conserved secondary structure features and sequences in miRNA transcripts that control Drosha activity and miRNA expression. Our results established the conservation of miRNA processing mechanisms and regulatory functions by Drosha and Dicer, greatly strengthened the evidence for the physiological consequences of relative specificity as well as demonstrated its evolutionary significance.

Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends

In plants, the RNase III-type enzyme Dicer-like 1 (DCL1) processes most microRNAs (miRNAs) from their primary transcripts called pri-miRNAs. Four distinct processing modes (i.e., short base to loop, sequential base to loop, short loop to base, and sequential loop to base) have been characterized in Arabidopsis, mainly by the Specific Parallel Amplification of RNA Ends (SPARE) approach. However, SPARE is a targeted cloning method which requires optimization of cloning efficiency and specificity for each target. PARE (Parallel Amplification of RNA Ends) is an untargeted method per se and is widely used to identify miRNA mediated target slicing events. A major concern with PARE in characterizing miRNA processing modes is the potential contamination of mature miRNAs. Here, we provide a method to estimate miRNA contamination levels and showed that most publicly available PARE libraries have negligible miRNA contamination. Both the numbers and processing modes detected by PARE were similar to those identified by SPARE in Arabidopsis. PARE also determined the processing modes of 36 Arabidopsis miRNAs that were unexplored by SPARE, suggesting that it can complement the SPARE approach. Using publicly available PARE datasets, we identified the processing modes of 36, 91, 90, and 54 miRNAs in maize, rice, soybean, and tomato, respectively, and demonstrated that the processing mode was conserved overall within each miRNA family. Through its power of tracking miRNA processing remnants, PARE also facilitated miRNA characterization and annotation.

Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

Whole-genome sequencing of follicular thyroid carcinomas reveal recurrent mutations in microRNA processing subunit DGCR8

Background The genomic and transcriptomic landscape of widely invasive follicular thyroid carcinomas (wiFTCs) and Hürthle cell carcinoma (HCC) are poorly characterized and subsets of these tumors lack information on genetic driver events. The aim of this study was to bridge this gap. Methods We performed whole-genome and RNA sequencing and subsequent bioinformatic analyses of 11 wiFTCs and 2 HCCs with a particularly poor prognosis, and matched normal tissue. Results All wiFTCs exhibited one or several mutations in established thyroid cancer genes, including TERT (n=4), NRAS (n=3), HRAS, KRAS, AKT, PTEN, PIK3CA, MUTYH, TSHR and MEN1 (n=1 each). MutSig2CV analysis revealed recurrent somatic mutations in FAM72D (n=3, in two wiFTCs and in a single HCC), TP53 (n=3, in two wiFTCs and a single HCC) and EIF1AX (n=3), with DGCR8 (n=2) as borderline significant. The DGCR8 mutations were recurrent p.E518K missense alterations, known to cause familial multinodular goiter via disruption of microRNA processing. Expression analyses showed reduced DGCR8 mRNA expression in FTCs in general, and the two DGCR8 mutants displayed a distinct miRNA profile compared to DGCR8 wildtypes. Copy number analyses revealed recurrent gains on chromosomes 4, 6 and 10, and fusiongene analyses revealed 27 high-quality events. Both HCCs displayed hyperploidy, which was fairly unusual in the FTC cohort. Based on the transcriptome data tumors amassed in two principal clusters. Conclusion We describe the genomic and transcriptomic landscape in wiFTCs and HCCs and identify novel recurrent mutations and copy number alterations with possible driver properties and lay the foundation for future studies.

TMOD-06. LOSS OF DICER COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE METASTATIC MEDULLOBLASTOMA

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.